Functional domains of a zinc metalloprotease from Vibrio vulnificus

Abstract

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS؉ was transformed into Escherichia coli DH5␣ for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37°C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. LineweaverBurk plot analysis indicated increased K m values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.Metalloproteases, in which zinc is an essential metal ion for catalytic activity, are elaborated by various human pathogenic bacteria, as well as by nonpathogenic ones (5). They can be classified into three families: thermolysins, serralysins, and neurotoxins (7). The members of the thermolysin family are synthesized as inactive precursors, with a large N-terminal propeptide acting as a specific inhibitor for the mature protease and/or an intramolecular chaperone to control the folding of the protease (10,14,20,24). Since the N-terminal propeptide is cleaved by autoproteolysis (9, 13, 24), a transformant carrying a cloned metalloprotease gene also produces a correctly processed metalloprotease (2, 13). On the other hand, studies of metalloproteases from vibrios (4,6,15) have demonstrated that, although these also belong to the thermolysin family, their precursors have C-terminal propeptides as well as N-terminal ones. The biochemical function of the Cterminal propeptide is currently unknown.Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia (8,22). Like other vibrios, this pathogen secretes the 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) since it belongs to the thermolysin family (18). VVP has been reported to have many biological functions: proteolytic degradation of a wide variety of host proteins, such as plasma proteins, involved in coagulation or complement action, induction of hemorrhagic tissue damage, and enhancement of vascular permeability through the generation of inflammatory mediators (18). Recently, ...

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

“…The 66 kDa protein corresponds to the VcpA zinc-metalloprotease whose gene was deleted in the DvcpA mutant. Isoforms of the VcpA metalloprotease may also occur in the secretome although it is not clear if isoforms have some proteolytic activity (Miyoshi et al, 1997;Park et al, 2008).…”

Generation of active fragments from human zymogens in the brady kinin-generating cascade by extracellular proteases from Vibrio vulnificus and V. parahaemolyticus

“…The recombinant V. vulnificus metalloprotease was used in the present experiments because of convenience of its purification (Miyoshi et al, 1997). The metalloprotease gene transformed into Escherichia coli is expressed by its own promoter, and the enzyme precursor produced is processed normally through autocatalytic removal of the N-terminal propeptide.…”

“…The recombinant V. vulnificus metalloprotease (45-kDa) was isolated from the periplasmic fraction of a transformant by ammonium sulfate precipitation followed by column chromatography on a HiLoad 16/10 Phenyl Sepharose column (Amersham Biosciences, Piscataway, NJ, USA) as described (Miyoshi et al, 1997). V. parahaemolyticus serine protease (50-kDa) was purified according to the procedures reported by Ishihara et al (2002).…”

SmcR and Cyclic AMP Receptor Protein Coactivate Vibrio vulnificus vvpE Encoding Elastase through the RpoS-dependent Promoter in a Synergistic Manner

“…The deduced gene product was predicted to be a 609-amino acid polypeptide, and the mature elastase is a 45-kDa protein consisting of 413 amino acids generated by the deletion of the N-terminal 196 amino acids. Using the mature protease purified from recombinant Escherichia coli, two functional domains, a 35-kDa Nterminal domain required for catalytic activity and 10-kDa domain required for attachment to substrate, were identified (7).…”